Evaluation of GABA Receptors of Ventral Tegmental Area in Cardiovascular Responses in Rat

Background: The ventral tegmental area (VTA) is well known for its role in cardiovascular control. It is demonstrated that about 20-30% of the VTA neurons are GABAergic though their role in cardiovascular control is not yet understood. This study is carried out to find the effects of GABA A and GABA B receptors on cardiovascular response of the VTA. Methods: Experiments were performed on urethane anesthetized male Wistar rats. Drugs were microinjected unilaterally into the VTA. The average changes in mean arterial pressure (MAP) and heart rate (HR) were compared between the case and the control groups using t test and with the pre-injection values using paired t test. Results: Microinjection of muscimol, a GABAA agonist (500, 1500 and 2500 pmol/100nl) into the VTA had no significant effect on MAP and HR compared with the saline group and pre-injection values. Injection of bicuculline methiodide (BMI, 100 and 200 pmol/100 nl), a GABAA antagonist, caused a significant increase in the MAP (11.1±1.95mmHg, P<0.5) and a decrease in HR (-32.07±10.2, P<0.01). Microinjection of baclofen a GABAB receptor agonist (500 or 1000 pmole/100 nl) and phaclofen a GABAB receptor antagonist (500 or 1000 pmole/100 nl) had no significant effects on MAP and HR. Conclusion: For the first time it was demonstrated that GABA system of the VTA inhibits the cardiovascular system through the activation of GABAA but not the GABAB receptors.


Introduction
The ventral tegmental area (VTA) is a part of mesolimbic dopamine system located in the medial portion of the ventral mesencephalon. The VTA can be divided into rostral and caudal sub-regions. The VTA is one of the dopaminergic (DA) origins in the central nervous system 1,2 and is well known for its role in cardiovascular regulation. [3][4][5] For instance, chemical stimulation of the VTA with L-glutamate decreases both arterial pressure (BP) and heart rate (HR) mediated by an inhibition of sympathetic but not the parasympathetic fibers. 6 Microinjection of substance P analogue, Dime-c7 into the VTA evokes dose-dependent increase in BP and HR. Intravenous pretreatment with dopamine D1 and D2 receptors antagonists markedly inhibited Dime-c7 effects as well as intravenous administration of vasopressin receptor antagonist. This suggests that an increase in vasopressin release is contributing to the rise in BP in response to Dime-c7 injection. 7,8 The pressor response of VTA to stimulation by substance P analogue is prevented by either bilateral electrolytic or chemical lesions of supraoptic nucleus of hypothalamus. 9 Miroinjection of tachykinin receptor agonists of NK1, NK2 and NK3 into the VTA are shown to increase BP and HR. These responses are inhibited by intravenous treatment with antagonist for D1 dopamine receptors and β1 adenoreceptor. 10 GABA injection into the VTA attenuated pressor response to administration of angiotensin II into lateral cerebral ventricle. 11 Injections of neurotensin into the VTA have no effect on mean arterial pressure and heart rate but significantly potentiated the pressor response to intravenous administration of vasopressin. This effect is blocked by intravenous pretreatment with raclopride. 8 The DA neurons comprise 70-80% of all neurons in the VTA 12 and the GABAergic neurons comprise about 20-30% of the VTA neurons. [12][13][14][15] The VTA receives GABAergic projection from ventral pallidum and nucleus accumbens 16 and ascend to nucleus accumbens 17 and prefrontal cortex. 18 The DA neurons receive GABAergic input from local GABAergic interneurons too. 19 Electrophysiological studies have shown that GABAB receptors in the VTA hyperpolarizes dopamine neurons. 2 Other evidence suggests that dopamine neurons are under tonic inhibition of the GABAergic afferents. 20 On the other hand, receptors of some neurotransmitters and neuropeptides are expressed on the local GABAergic neurons. For example, acethylcholine through its muscarinic receptor increases the frequency of spontaneous inhibitory postsynaptic current (IPSC's) in GABAergic neurons. 19 Activation of muscarinic receptors presynaptically inhibits the amplitude of GABAergic synaptic currents. 19,21 Considering the above, this study was performed to find the role of GABAergic system of VTA in cardiovascular regulation and to assess the role of each GABA receptor subtype by microinjection of selective agonists and antagonists into the VTA in urethane anesthetized male rats.

Subjects and Surgery
The experiments were performed on 62 male Wistar rats (200-300 g) in accordance and approval by the Animal Use and Care Committee of Shiraz University of Medical Sciences. The animals were randomly divided into six groups and were anesthetized with urethane (1.4 g/kg, ip) and supplementary doses (0.7 g/kg) were given as required. The trachea was cannulated to ease ventilation and their body temperature was maintained at 37±1° C by an animal temperature controller (Narco-Bio. System, USA). The femoral artery was cannulated with polyethylene catheter (PE-50) filled with heparinized saline and the catheter was connected to a pressure transducer (Harvard). The arterial pressure and heart rate were continuously recorded by both a Harvard polygraph and a computer program (written by Dr A. Nasimi). In some experiments, femoral artery was connected to a ML T844 pressure transducer coupled to a pre-amplifier (FE221 bridge amplifier) connected to power lab 4/35 data acquisition system (model PL3504ADinstruments). The animals were placed in prone position in a stereotaxic frame (Stoelting, USA) and two small holes were drilled through the parietal bone over the VTA.

Drugs Microinjection
Unilateral microinjection of drugs was performed by a micropipette with 35-45 µm internal diameter. Micropipette tips were positioned in the VTA according to a stereotaxic atlas of the rat brain. 22 The stereotaxic coordinates of the VTA were explored from 4.6 to 5.2 mm caudal to bregma, midline to 0.7-1.3 mm lateral and 8.2-8.4 to ventral from the bregma. Pressure injection was performed by a pressurized air pulse applicator and the volume of injection was measured by direct observation of the fluid meniscus in the micropipette by using a microscope fitted with an ocular micrometer (made in U.W.O, Canada). The injection volume of all the drugs was 100nl.
All drugs except phaclofen were dissolved in saline. Phaclofen was dissolved in 4% of HCL 0.1 N and 96% of saline, and the PH was raised to seven by adding NAOH. The injection sites were 100 µm apart and 1-3 injections were made in each animal on both sides.

Experimental Groups
The experiments for studying the GABAergic system of VTA consisted of 6 groups as follow: -Microinjection of saline or vehicle of phaclofen (n=9 rats, injections=12) as the control groups. All procedures were exactly the same as the experimental groups but instead of drugs, the vehicle was injected into the VTA.

Histology
At the end of each experiment, the animal was sacrificed by a high dose of the anesthetic and then perfused transcardially with 100 ml of 0.9% saline followed by 100 ml of 10% formalin. The brain was removed and stored in 10% formalin for at least 24-h. Frozen serial transverse sections (50 micron) of the brain were cut and stained with cresyl violet 1%. The injection sites were determined according to a rat brain atlas 22 under microscope light.

Data Analysis
The results are expressed as mean±standard error of mean (SE). Initially Kolmogorov-Smirnov normality test was performed on data and all were found to be normal.
The mean of the maximum changes of the heart rate and the mean arterial pressure were compared with those of the control group using independent t-test and with the pre-injection values using paired t-test. A P<0.05 was taken to indicate statistical significance.

Cardiovascular Responses to BMI Microinjected into the VTA
Different doses of bicuculline methiodide (BMI), a GABA A antagonist, were injected into the VTA of anesthetized rats (basal MAP=93.8±4.8 and basal HR=369.6±7.9). The injections were; 100pmol/100nl (∆MAP=11.1±1.95mmHg, ∆HR=-32.07±10.2 bpm, n=14 rats, 28 injections) and 200 pmol/100 nl (15.4±5.8 mm Hg, ∆HR =-113.6±32.6 beats/min n=10 rats, 19 injections). Figure 3 shows a sample of the arterial pressure and HR tracings before and after the injection of 200 pmol/100 nl BMI. As shown, BMI increased MAP and decreased HR in the majority (70%) of the cases. In this group, the range of the MAP change was from 5 to 57 mm Hg and the range of the bradycardia was from -10 to -290 beats/ min. The variations reached a peak at 3 to 10 minutes and returned to baseline at most in 30 minutes. In some experiments, high doses produced higher hypertension and bradycardia which returned to baseline after around 1h. Compared with the baseline values, BMI caused a peak significant increase in the MAP (paired t test, P<0. 05) and significant decrease in the HR (paired t-test, P<0.001). Also the maximum MAP and HR changes were significantly (independent t test, P<0.01) different from that of the saline group (figure 2).
A photomicrograph of the VTA is shown in figure  6 that shows two injection sites in the VTA. The distribution of the injection sites is shown in figure 7.

Discussion
In the present study we demonstrated that GABAergic neurons play a major role in the cardiovascular responses of the VTA. Two regions of the VTA are reported to contain glutamic acid decarboxylase (GAD-an enzyme critical for GABA synthesis). 12 The regions in this experiment are the same regions containing GAD enzymes. These areas correspond to the regions in our experiments (figures 6 and 7). The most responsive site was found to be in the rostral VTA 4.7-5.0 mm caudal to bregma which corresponds with GAD positive neurons. 12 Microinjection of bicuculline, a GABA A receptor antagonist, produced dose dependent pressor responses concomitant with marked bradycardia (figures 2 and 3). However, different doses of muscimol, a GABA A receptor agonist, had no significant effect on blood pressure or heart rate. The pressor effect of BMI as well as insignificant response to muscimol injections implies that there should be a tonic release of GABA inhibiting the cardiovascular function via GABAA receptor. This is either by interneurons which are dispersed among the dopamine neurons 13,19 or via GABAergic inputs to the VTA such as direct inputs from the nucleus accumbense, 19,23 ventral pallidum, 23,24 reticulate parts of substantia nigra 13,25 and the bed nucleus of the stria terminalis. 26 GABAergic neurons in some of these nuclei exert the same response as the VTA, for example the GABAergic system of the bed nucleus of the stria terminalis is tonically active and decreases the arterial pressure and heart rate. 27 There is a GABAergic projection from VTA/SN to ventrolateral periaquductal gray region (PVGVL) and dorsal raphe nucleus (DR). 28 Microinjection of GABA blocking agent, picrotoxin   into the PVGVL/DR eliminated the cardiovascular depressor response by glutamate stimulation of VTA/SN. 28 In an electrophysiological study that activation of GABA A receptor mediated post synaptic current (IPSC s ) were recorded from dopaminergic neurons of the VTA in rats. 29 Also the activation of presynaptic GABAA receptor facilitates further GABA release in VTA. 29 These results provide the evidence that GABA mediated its inhibition through GABA A receptors via both dopaminergic and nondopaminergic neurons within the VTA. 30 Microinjection of phaclofen a GABA B receptor antagonist, and baclofen a GABA B receptor agonist had no significant effect on AP and HR. Lack of response to phaclofen, demonstrated that the GABA cardiovascular responses are not mediated through GABA B receptors. However, this result cannot rule out the presence and other possible effects of GABA B receptors. For example, GABAB receptors are present on GABAergic terminals synapsing on dopaminergic neuron of the VTA; activation of these receptors presynaptically attenuates the release of GABA in the VTA. 29 Furthermore, stimulation of GABAB receptors in the VTA blocked opiate-induced motor stimulation and motor sensitization by inhibiting the nucleus accumbens neurons. 16 In addition, GABAB receptor of the VTA is involved in expression of morphine-induced conditioned place preference in rats. 31 The bradycardic response to increase of BP implies that the VTA might be a part of the central baroreflex pathways. Experimental evidence has suggested that the DA neurons may be regulated by input from arterials baroreceptors. 3,32,33 This proposition is based on the finding that denervation of arterial baroreceptors caused decrease in dopamine content and in the activity of dopamine biosynthetic enzyme tyrosine hydroxylase in the striatum part of the VTA. 34 Some evidence suggests that dopamine neurons are under tonic inhibition of the GABAergic afferents; 20 the GABAergic neurons of the VTA might be responding to baroreceptor input.
In summary, for the first time it was demonstrated that GABAergic neurons of the VTA are involved in cardiovascular responses through the activation of GABAA but not GABAB receptors. Theses neurons are tonically active under normal conditions.